Project 2 will provide add new insight into pharmacologic inhibition of FLT3 in patients treated with PKC412 on clinical trials in Project 1 (Specific Aim 1), In addition (specific Aim 2), the Project will characterize mechanisms of resistance of FLT3 to small molecule inhibitors such as PKC412. These data will have important clinical implications, both in assessing response to FLT3 inhibitors in vivo and as a platform for developing strategies to overcome resistance. In specific aim 3 the Project will screen for novel tyrosine kinase mutations in a genome wide high-throughput screen to include (a) DMA sequencing screen using our existing platform, (b) screening for kinase mutations using high density oligonucleotide array CGH and , (c) characterizing transforming properties in cell culture systems. The gene discovery based platform in Specific Aim 3 could lead to identification of potential targets for therapy. Collaboration with Project 1 has begun with respect to characterizing responders to PKC412. The first FLT3 wild type patient found by Dr. Gilliland to have FLT3 over expression as a possible basis for response to the drug was treated at M.D. Anderson .Using a test set of 42 MDS cases, proof-of-principle has been developed for the strategy of high-throughput screening for activating tyrosine kinase mutations. Although analysis of all exons of all kinases has not been completed, 2 novel and promising mutations have been identified one of these is a novel mutation in FLT3, and another in EphA2. In work conducted since the site visit we have successfully developed a platform for high-density oligoarray comparative genomic hybridization for detection of 5' flanking deletions that activate tyrosine kinases. We have also developed a strategy to screen for similar deletions across the entire kinome comprised of -90 fully annotated tyrosine kinases, and will apply this to screen MDS patients for activating mutations.